• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.5
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• pp.619-624
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• 2011

Tea extract (TE) has been shown to have anti-tumor properties in a wide variety of experimental systems. We evaluated green tea extract (GTE) as a biochemical modulator for the antitumor activity of cisplatin and doxorubicin in the treatment of human lung cancer A549 cells. Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and two antibiotics (100 units/mL penicillin and $100\;{\mu}g$/mL streptomycin). Two types of TE, epigallocatechin galate (EGCG) and GTE, were used in this experiment. The cells were seeded at $1{\times}10^4$ cells/well in the RPMI-1640 media with or without TE ($100\;{\mu}g$/mL) and then treated with different concentrations of doxorubicin ($0{\sim}14\;{\mu}g$/mL) or cisplatin ($0{\sim}35\;{\mu}g$/mL). After incubation in 5% $CO_2$ at $37^{\circ}C$ for 24 hr, cell viability was determined with a MTT assay. We used a Western blot to detect the influence of EGCG and GTE on the expression of p53 and caspase-3 genes in the A549 cells. A549 cell viability decreased to 15% with a $10\;{\mu}g$/mL concentration of cisplatin, and to 21% with a $8\;{\mu}g$/mL concentration of doxorubicin, as measured with the MTT assay. However, pre-treatment of the cells with EGCG ($100\;{\mu}g$/mL) or GTE ($100\;{\mu}g$/mL) resulted in decreased cell viability with $6\;{\mu}g$/mL of cisplatin and $4\;{\mu}g$/mL of doxorubicin. There was no apparent change in cell viability between EGCG or GTE administration in cisplatin- or doxorubicin-induced cytotoxicity in A549 cells. The levels of p53 and caspase-3 in the A549 cells increased with both EGCG and GTE treatment. We found that GTE could potentially affect cisplatin- or doxorubicin-induced cytotoxicity of A549 cells, which may be useful in the chemotreatment of cancer.

• Journal of Life Science
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• v.26 no.7
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• pp.847-854
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• 2016

Cordycepin, a derivative of the nucleoside adenosine, is one of the active components extracted from fungi of genus Cordyceps, and has been shown to have many pharmacological activities. In this study, we investigated the effects of cordycepin on proliferation and apoptosis of human gastric cancer AGS cells, and its possible mechanism of action. Treatment of cordycepin resulted in significant decrease in cell viability of AGS cells in a concentration-dependent manner. A concentration-dependent apoptotic cell death was also measured by agarose gel electrophoresis and flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled cordycepin treatment resulted in an enhanced expression of tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5 and Fas ligand. Furthermore, up-regulation of pro-apoptotic Bax, and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL expression were also observed in cordycepin-treated AGS cells. These were followed by activation of caspases (caspase-9, -8 and -3), subsequently leading to poly (ADP-ribose) polymerase cleavage. Taken together, these findings indicate that cordycepin induces apoptosis in AGS cells through regulation of multiple apoptotic pathways, including death receptor and mitochondria. Although further mechanical studies are needed, our results revealed that cordycepin can be regarded as a new effective and chemopreventive compound for human gastric cancer treatment.

• Archives of Pharmacal Research
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• v.24 no.2
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5887-5895
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• 2012

Background: To determine the effect of essential oil obtained from a traditionally used medicinal plant Tridax procumbens L, on lung metastasis developed by B16F-10 melanoma cells in C57BL/6 mice. Materials and Methods: Parameters studied were toxicity, lung tumor nodule count, histopathological features, tumor directed capillary vessel formation, apoptosis and expression levels of $P^{53}$ and caspase-3 proteins. Results: In vitro the MTT assay showed cytotoxicity was found to be high as 70.2% of cancer cell death within 24hrs for $50{\mu}g$. In vivo oil treatment significantly inhibited tumor nodule formation by 71.7% when compared with untreated mice. Formation of tumor directed new blood vessels was also found to be inhibited to about 39.5%. TUNEL assays also demonstrated a significant increase in the number of apoptotic positive cells after the treatment. $P^{53}$ and caspase-3 expression was also found to be greater in the essential oil treated group than the normal and cancer group. Conclusions: The present investigation showed significant effects of the essential oil of Tridax procumbens L in preventing lung metastasis by B16F-10 cell line in C57BL/6 mice. Its specific preventive effect on tumor directed angiogenesis and inducing effect on apoptosis warrant further studies at the molecular level to validate the significance of Tridax procumbens L for anticancer therapy.

• Journal of Pharmacopuncture
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• v.20 no.1
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• pp.29-35
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• 2017

Objectives: Aging is an unconscious and gradual process that can lead to changes in biological systems. Induction of oxidative stress and apoptosis, hepatotoxicity and neurotoxicity are involved in the aging process. Regarding the antioxidant property of black seed oil, the aim of this study was to evaluate the anti-aging effect of Nigella sativa (N. sativa) oil on d-galactose-induced aging in mice. Methods: For induction of aging, D-galactose (500 mg/kg, subcoutaneously SC) was administrated to male mice for 42 days. Animals were treated with D-galactose alone or with b lack seed oil (0.1, 0.2, 0.5 mL/kg, intraperitoneally (ip)). Additionally, vitamin E (200 mg/kg) was used as a positive control. At the end of treatment, the malondialdehyde (MDA) and the glutathione (GSH) contents in brain and liver tissues were measured. Also, enzymes in serum, including aspartate aminotransferase (AST) and alanine amino transferase (ALT), were determined. The levels of the proteins Bax, Bcl2, caspase-3 (pro and cleaved) in brain and liver tissues were evaluated. Results: Administration of D-galactose (500 mg/kg, SC) for 42 days increased serum levels of ALT and AST, as well as the MDA content, in brain and liver tissues, but decreased the GSH content. Additionally, the levels of apoptotic proteins, including Bax, procaspase-3 and caspase-3 cleaved, were markedly increased. N. sativa oil (0.1 and 0.2 mL/kg) diminished the levels of the biochemical markers ALT and AST. Administration of black seed oil (0.1, 0.2 and 0.5 mL/kg) reduced lipid peroxidation and at doses 0.1 and 0.2 mL/kg significantly recovered the GSH content. The oil decreased Bax/Bcl2 levels and at 0.1 mL/kg down-regulated the expressions of caspase-3 (pro and cleaved) proteins in brain and liver tissues. Conclusion: Through its antioxidant and anti-apoptosis properties, black seed oil exhibited an anti-aging effect in a model of aging induced with D-galactose.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.950-956
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• 2002

The antitumor activity of the insect pathogenic fungus Paecilomyces japonica has been attributed to apoptotic cell death. However, the mechanism underlying the induced apoptosis has not yet been elucidated. In this study, we for the first time show that mitochondria-dependent caspase-3 activation were associated with the apoptotic activity of P. japonica in human acute leukemia Jurkat T cells. When Jurkat T cells were treated with the ethyl acetate extract of P japonica at concentrations ranging from $2-6{\mu}g/ml$, apoptotic cell death. accompanied by several biochemical events such as caspase-9 activation, caspase-3 activation, degradation of poly (ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation, was induced in a dose-dependent manner. In addition, the release of cytochrome c from mitochondria was detected. Under these conditions, the expression of Fas and Fas-ligand (FasL) remained unchanged. Ethyl acetate extract-induced mitochondrial cytochrome c release, caspase-3 activation, PARP cleavage, and apoptotic DNA fragmentation were suppressed by the ectopic expression of Bcl-2, which is known to block mitochondrial cytochrorme c release. Accordingly, these results demonstrate that P. japonica-induced apoptotic cell death is mediated by a cytochrome c-dependent caspase-3 activation pathway that can be interrupted by Bcl-2.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7919-7923
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• 2014

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown to inhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticancer activity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPD and cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and Annexin V-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2 and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetric assay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an $IC_{50}$ value of $33.3{\mu}M$ at 24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysis in cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and 30.5% in MCF-7 cells treated with 0, 15, 30 and $60{\mu}M$ of 20(S)-PPD, respectively. Moreover, 20(S)-PPD could induce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression. These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD was blocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death. In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death by a caspase-mediated apoptosis pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3289-3294
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• 2016

Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dose-dependent, measured by MTT assay. Naringin-treated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and flow cytometer was reduced concentration-dependently, which indicated influence on the mitochondrial signaling pathway. Caspase-3, -8 and -9 activities were enhanced as evidenced by colorimetric detection of para-nitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringin-treated HepG2 cell apoptosis. The expression levels of pro-apoptotic Bax and Bak proteins were increased whereas that of the anti-apoptotic Bcl-xL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase-8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondria-mediated activation of caspase-9 and caspase-8-mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.

• Development and Reproduction
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• v.12 no.3
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• pp.297-303
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• 2008

BCL-2 family members are essential protein for the regulation of cell death and survival consisting both antiapoptotic and pro-apoptotic proteins. In the present study, we designed and cloned a new apoptotic molecule MCL-1ES BH3M coding a modified protein of MCL-1L. Compared to MCL-1L protein, MCL-1ES BH3M lacks the PEST motifs known to be involved in MCL-1L protein degradation and has seven mutated residues in BH3 domain critical for dimerization with BCL-2 family members. Overexpression of MCL-1ES BH3M induced death of different cells, and its cell killing effect was not blocked by forced expression of the pro-survival protein MCL-1L. Expression of MCL-1ES BH3M protein led to the activation of caspase 9 and caspase 3, suggesting apoptotic cell death, and confocal fluorescent microscopic analyses showed that MCL-1ES BH3M was partially localized in mitochondria. In conclusion, we reported a new apoptotic molecule and determined its cell death activity in cells.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.12
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• pp.1708-1716
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• 2016

The Akt/mammalian target of the rapamycin (mTOR) pathway is activated in the majority of human cancers. Activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy. In this study, we evaluated the apoptotic effect of ethanol extract of Artemisia annua L. through down-regulation of Akt signal pathways and the mitochondrial pathway in hepato-carcinoma cells (HepG2). A. annua extract is known as a medicinal herb that is effective against cancer. We evaluated anti-proliferative activity by MTT-based viability assay and apoptotic effect by Annexin-V/PI staining, mitochondrial membrane potential (MMP), and caspase-3/7 activity as determined by flow cytometry. A. annua treatment led to loss of MMP, resulting in cytochrome c-inducible activation of caspase-3/7. Treatment with A. annua extract reduced activities of Akt/mTOR/anti-apoptotic proteins (such as Bcl-2 and $Bcl-X_L$), leading to increased activation of tumor suppressor p53 and pro-apoptotic proteins (such as Bax and Bak). We applied LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR) to determine the relationship between signal transduction of proteins associated with apoptosis. LY294002 and rapamycin significantly reduced cell viability and increased apoptosis. These results indicate that Bcl-2 and caspase-3 are key regulators in A. annua extract-induced apoptosis in HepG2 cells and are controlled through the Akt/mTOR signaling pathway.